Role of Pim-1 in Smooth Muscle Cell Proliferation*

The proliferation of vascular smooth muscle cells (VSMCs) and alterations of their phenotype are implicated in the pathogenesis of atherosclerosis. Arterial wall injury induces the expression of proto-oncogenes, leading to the proliferation of VSMCs. In particular, c-Myc and c-Myb play a central role in cell cycle progression and are essential for VSMC replication. The proto-oncogene Pim-1 cooperates with c-Myc and enhances the transcriptional activity of c-Myb in hematopoietic cells, suggesting that Pim-1 is involved in cell cycle regulation. The aim of this study was to examine the possible involvement of Pim-1 in VSMC proliferation. Pim-1 was substantially induced in neointimal VSMCs of bal-loon-injured rat carotid arteries, and in vivo infection with a dominant negative Pim-1-expressing adenovirus (Ad-DN-Pim-1) markedly suppressed neointima formation and cell cycle progression in the balloon-injured arteries. In cultured VSMCs, treatment with serum or H 2 O 2 induced Pim-1 Biotechnol-ogy) and then incubated with a biotinylated secondary antibody from the Vectastain Elite ABC kit (Vector). Positive signals were visualized by incubating with 3, 3 (cid:2) -diaminobenzidine peroxidase substrate (Vec- tor). For the double immunostaining for Pim-1 and (cid:3) -smooth muscle actin, anti- (cid:3) -smooth muscle actin mouse monoclonal antibody (1A4) (DAKO) was used as a primary antibody, and (cid:3) muscle actin was visualized with red. To confirm the expression of in arteries, common artery segments were embedded in frozen media and sec- tioned (5 for fluorescent microscopy examination. Blot Analysis— Fifty extracted from VSMCs were subjected to SDS-PAGE and with a specific antibody or anti- a peroxidase-conju-gated antibody Detection was and results were quantified by densitometry. Analysis— analysis of a of stained with and was performed with an automated soft-ware by a single investigator who blinded to To evaluate thickening of the neointima, the areas encroached by the external elastic lamina (EEL area), the internal elastic lamina (IEL and the area were measured. The the and neointima-to- were calculated as follows:

The proliferation of vascular smooth muscle cells (VSMCs) 1 and alterations of their phenotype are implicated in the patho-genesis of atherosclerosis and restenosis after the angioplasty of vascular lesions (1)(2)(3). Injury of the arterial wall initiates the synthesis and release of numerous mitogens and the rapid induction of proto-oncogenes, leading to the proliferation of VSMCs. In particular, c-Myc and c-Myb have been found to play a central role in cell cycle progression and to be essential for VSMC replication, which has been demonstrated by various in vitro and in vivo studies (4 -7). Furthermore, the downregulation of these molecules has been reported to interfere with VSMC proliferation (8 -12). Cyclin/cyclin-dependent kinases are also important regulators of cell proliferation; it has been shown that the cyclin-dependent kinase inhibitor p21 blocks VSMC proliferation and inhibits neointima formation after injury (13,14).
Pim-1 is a proto-oncogene that encodes a serine/threonine kinase whose expression is associated with the survival and proliferation of hematopoietic cells (15,16). Its expression is induced by a variety of cytokines, growth factors, and mitogens (17)(18)(19), suggesting that Pim-1 may be an important intermediate in signal transduction. In hematopoietic cells, Pim-1 cooperates with c-Myc (20,21) and enhances the transcription activity of c-Myb (22,23). It has also been shown that Pim-1 activates the cyclin-dependent kinase inhibitor p21 (24) and that the antiapoptotic factor Bcl-2 is regulated downstream of Pim-1 (25), suggesting that Pim-1 is involved in cell cycle regulation and apoptosis in hematopoietic cells. In addition, functional analysis by RNA interference in embryonic stem cells has recently demonstrated that Pim-1 is required for their differentiation into endothelial cells and VSMCs (26). In this study, we show that Pim-1 expression is observed in ballooninjured rat carotid arteries and human coronary arteries and that dominant negative Pim-1 markedly suppresses VSMC proliferation in the balloon-injured model and in a cell culture system. chloride gradient followed by extensive dialysis. The titer of the virus stock was assessed by a plaque formation assay using 293 cells and expressed as plaque-forming units. We also prepared two control adenoviruses, namely Ad-LacZ, expressing bacterial ␤-galactosidase, and Ad-GFP, expressing green fluorescent protein.
Rat Carotid Artery Balloon Injury Model-Male Sprague-Dawley rats (weighing 300 -350 g at 9 to10 weeks of age; Clea Japan, Tokyo Japan) anesthetized with pentobarbital (50 mg/kg intraperitoneal) were subjected to balloon angioplasty of the left common carotid artery by dilatation with a balloon catheter (2F Fogarty; Baxter) three times, and then 50 l of Ad-DN-Pim-1 or a control virus expressing green fluorescent protein (Ad-GFP) (1 ϫ 10 9 plaque-forming units/ml) was injected into a 10-mm segment of the distal common carotid artery over a period of 20 min by using a 24-gauge intravenous catheter. The solution was then retrieved, the catheter removed, and blood circulation restored. Fourteen days after balloon injury, rats were killed, and the right and left common carotid arteries were excised and fixed with 4% paraformaldehyde for morphometric and immunohistochemical analysis. Five arterial segments 2 mm in length were obtained, embedded in paraffin, and cut into 5-m-thick slices. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Osaka University Graduate School of Medicine.
Immunohistochemistry-Immunostaining for Pim-1 and the proliferating cell nuclear antigen (PCNA) was performed as follows. After blocking with 10% normal serum (Vector), sections were incubated overnight at 4°C with a 1:100 dilution of anti-Pim-1 goat polyclonal IgG antibody (C-20), anti-PCNA mouse monoclonal antibody (PC 10), or anti-c-Myc mouse monoclonal antibody (9E10) (Santa Cruz Biotechnol-ogy) and then incubated with a biotinylated secondary antibody from the Vectastain Elite ABC kit (Vector). Positive signals were visualized by incubating with 3, 3Ј-diaminobenzidine peroxidase substrate (Vector). For the double immunostaining for Pim-1 and ␣-smooth muscle actin, anti-␣-smooth muscle actin mouse monoclonal antibody (1A4) (DAKO) was used as a primary antibody, and ␣-smooth muscle actin was visualized with fuchsin red. To confirm the expression of GFP in treated arteries, representative common carotid artery segments were embedded in frozen section media (OCT medium, Tissue-Tek) and sectioned (5 m) for fluorescent microscopy examination.
Western Blot Analysis-Fifty micrograms of protein extracted from cultured VSMCs were subjected to SDS-PAGE and Western blotting with a specific primary antibody (anti-Pim-1 (C-20), anti-c-Myc (9E10), or anti-␤-galactosidase (Sigma)) and a horseradish peroxidase-conjugated secondary antibody (Zymed Laboratories Inc.). Detection was performed by enhanced chemiluminescence (PerkinElmer Life Sciences), and the results were quantified by densitometry.
Morphometric Analysis-Morphometric analysis of a cross-sectional section of carotid arteries stained with hematoxylin and eosin was performed with an automated computer-based image-analyzing software package (NIH image) by a single investigator (K. S.) who was blinded to the treatment protocol. To evaluate thickening of the neointima, the areas encroached by the external elastic lamina (EEL area), the internal elastic lamina (IEL area), and the lumen area were measured. The medial area, the neointimal area, and the neointima-tomedia ratio were calculated as follows: medial area ϭ EEL area Ϫ IEL area; neointimal area ϭ IEL area Ϫ lumen area; neointima-to-media ratio ϭ neointimal area/medial area.
Terminal Deoxynucleotidyltransferase-mediated dUTP Nick End-labeling (TUNEL) Assay-Perfusion-fixed, paraffin-embedded tissues were stained for apoptotic nuclei with an in situ apoptosis detection Kit (Takara). In brief, after treatment with proteinase K, slides were incubated with a TUNEL reaction mixture, treated with converter-peroxidase solution, and exposed to diaminobenzidine black nickel chromagen. Slides were counterstained with hematoxylin and analyzed by a light microscopy for total and TUNEL-positive cells.
Culture and Stimulation of VSMCs-VSMCs were grown from explants of 4-week-old male Sprague-Dawley rat aortic arteries and maintained in Dulbecco's modified Eagle's medium supplemented with penicillin, streptomycin, glutamine, nonessential amino acids, and 20% (primary cultures) or 10% (passaged cells) fetal calf serum (FCS). The VSMCs were used for experiments at passages Ͻ5. The identity of the smooth muscle cells was confirmed by immunocytochemistry for smooth muscle cell ␣-actin. Synchronous populations of quiescent cells were obtained by placing cultures in media that contained 0.5% FCS for 48 h. Cells were then infected with adenovirus (Ad-DN-Pim-1 or Ad-LacZ) diluted in starvation medium at a multiplicity of infection of 100 for 1 h. Cells were then stimulated with the addition of serum mitogens (10% FCS in Dulbecco's modified Eagle's medium) or H 2 O 2 (5 or 10 M).
Immunocytochemistry-VSMCs were cultured onto Lab-Tek tissue culture chamber slides (Invitrogen), fixed in ice-cold methanol, quenched using bovine serum albumin, and incubated with a specific primary antibody (anti-Pim-1 (C-20) or anti-c-Myc (9E10 epitope)) for 2 h. After three washes in phosphate-buffered saline, cells were incubated with fluorescein isothiocyanate-conjugated secondary antibody for 60 min. After three washes in phosphate-buffered saline, stained cells were examined using fluorescence microscopy.
Northern Blot Analysis-Total RNA was isolated from VSMCs before and after treatment with 200 M H 2 O 2 . Twenty micrograms of the total RNA was size-fractionated by 1.2% agarose gel electrophoresis and transferred to a nylon membrane. The probes used were 32 P-labeled Pim-1 cDNA (1 kb; EcoRI-XhoI fragment). After hybridization with the labeled probes at 42°C, the membrane was washed twice with 2ϫ FIG. 1. Pim-1 expression in the neointima of balloon-injured rat carotid arteries. Fourteen days after balloon injury, neointima formation is clearly visible, and Pim-1 expression is substantially induced in the neointima of injured arteries (right) but not in the control uninjured artery (left). IEL, internal elastic lamina.

FIG. 2. Adenovirus-mediated expression of GFP and DN-Pim-1 in balloon-injured rat carotid arteries.
Ad-DN-Pim-1 and Ad-GFP were prepared, and 50 l of each adenovirus (1 ϫ 10 9 plaque-forming units/ml) was delivered to balloon-injured rat carotid arteries. A, top, representative balloon-injured rat carotid artery 14 days after Ad-GFP infection. As revealed by the GFP signal in this fluorescence micrograph, many neointimal cells are infected after exposure to the adenovirus. Bottom, immunostaining for DN-Pim-1 in balloon-injured rat carotid arteries infected with or without Ad-DN-Pim-1. DN-Pim-1 expression is observed in neointimal smooth muscle cells after exposure to Ad-DN-Pim-1. B, DN-Pim-1 protein expression after Ad-DN-Pim-1 infection. Fourteen days after balloon injury, total protein was obtained from uninfected, Ad-GFP infected, or Ad-DN-Pim-1 infected carotid arteries, and Western blot analysis was performed with an antibody for Pim-1. sodium chloride-sodium citrate (1 ϫ SSC, 15 mM sodium citrate and 150 mM NaCl, pH 7.5), which contained 0.1% sodium dodecyl sulfate at 50°C for 60 min and was then washed with 0.2ϫ SSC and 0.1% SDS at 50°C for 30 min. Autoradiography was performed using an intensifying screen at Ϫ80°C, and the exposure time was varied so that the band intensity was kept within the linear range.
Determination of DNA Synthesis-Relative rates of DNA synthesis were assessed by the determination of [ 3 H]thymidine incorporation into trichloroacetic acid-precipitable material. Rat VSMCs (5 ϫ 10 3 cells) were plated per well of a 24-well plate in growth medium. Quiescent cells were then stimulated with serum or H 2 O 2 . The cells were exposed to [ 3 H]thymidine (Amersham Biosciences) at a concentration of 1 Ci/ml for the last 4 h of the 24-hour stimulation period. The cells were then incubated with 5% trichloroacetic acid at 4°C for 20 min, dissolved in 1 N NaOH at 37°C for 20 min and then neutralized. Radioactivity was determined by liquid scintillation counting. Each experiment was performed in triplicate and repeated on VSMCs isolated from several separate donors.
Flow Cytometric Analysis of Cell Cycle Stage-After detachment with 0.25% trypsin, VSMCs were resuspended, fixed in 70% ethanol, stained in a solution of phosphate-buffered saline, propidium iodide (50 g/ml), and RNase A (500 g/ml), and measured with a FACScan apparatus (BD Biosciences). The data were analyzed for cell cycle distribution using the ModFitLT program.
Thoracic Aortas and Coronary Arteries from Individuals at Autopsy-Thoracic aortas were obtained at autopsy from three individuals aged 73 (case 1), 45 (case 2), and 35 years (case 3) whose causes of death were acute myocardial infarction (case 1 and case 2) and dilated cardiomyopathy (case 3). Coronary arteries were obtained at autopsy from three individuals aged 62 (case 4), 83 (case 5), and 32 years (case 6) whose causes of death were acute myocardial infarction, aortic aneurysm, and primary pulmonary hypertension, respectively. The study protocol was approved by the hospital ethics committee, and informed consent was obtained from each patient's bereaved relative.
Statistical Analysis-All values are expressed as mean Ϯ S.D. Data between two groups were compared by a two-tailed unpaired Student's t test, and data between more than two groups were compared by a one-way analysis of variance followed by Scheffe's test. A value of p Ͻ 0.05 was considered to be statistically significant.

Pim-1 Expression in Balloon-injured Rat Carotid
Arteries-To examine the possible involvement of Pim-1 in the progression of atherosclerosis, immunohistochemical staining for Pim-1 was performed in balloon-injured rat carotid arteries. We first examined whether the expression of Pim-1 is induced in the arterial wall. As shown in Fig. 1, 14 days after balloon injury neointima formation was clearly visible, and Pim-1 expression was substantially induced in the neointima of injured arteries but not in the control uninjured artery. To the best of our knowledge, this is the first report on the expression of Pim-1 in the vascular wall.
Inhibition of Pim-1 Activity Attenuates Neointima Formation-To examine the possible role of Pim-1 in smooth muscle cell proliferation, we evaluated the effect of the suppression of Pim-1 activity on neointima formation after balloon injury. For this purpose, we prepared adenovirus-expressing dominant negative Pim-1 (Ad-DN-Pim-1) and control adenovirus-expressing GFP (Ad-GFP) and delivered 50 l of each adenovirus (1 ϫ 10 9 plaque-forming units/ml) to balloon-injured rat carotid arteries. Fig. 2A, top, shows a representative balloon-injured rat carotid artery 14 days after Ad-GFP infection. As revealed by the GFP signal in this fluorescence micrograph, many neointimal cells were infected after exposure to the adenovirus. Immunostaining of balloon-injured arteries after Ad-DN-Pim-1 infection revealed that DN-Pim-1 expression was induced in neointimal smooth muscle cells (Fig. 2A, bottom). Expression of the DN-Pim-1 protein was also confirmed by Western blotting (Fig. 2B). Fig. 3A shows a representative rat carotid artery 14 days after balloon injury. In the injured artery exposed to saline or Ad-GFP, a significant concentric neointima was observed with the medium clearly defined by the internal and external elastic laminae (neointimal cross-sectional area for saline treated was 0.12 Ϯ 0.05 mm 2 , and for Ad-GFP treated it was 0.12 Ϯ 0.06 mm 2 ) (n ϭ 6). Overexpression of DN-Pim-1 resulted in marked suppression of the neointimal cross-sectional area (0.04 Ϯ 0.02 mm 2 ) (n ϭ 6). The intima/medium ratio in arteries exposed to Ad-DN-Pim-1 (0.29 Ϯ 0.10) was also significantly less than in arteries exposed to either saline (0.88 Ϯ 0.37) or Ad-GFP (0.85 Ϯ 0.26). There was no significant difference in the medial area (Fig. 3B). In addition, to elucidate the mechanism for the suppression of neointima formation in Ad-DN-Pim-1-treated arteries we examined the effect of DN-Pim-1 overexpression on the cell proliferation rate. Many PCNA-positive cells were observed in Ad-GFPtreated arteries but not in Ad-DN-Pim-1-treated arteries (Fig. 4). In addition, because it has recently reported that Pim-1 could regulate apoptosis (27), we performed a TUNEL assay to examine whether the effects of DN-Pim-1 in our present study relate to an increase of apoptosis. In contrast to the decrease in the number of PCNA-positive cells, the number of apoptotic cells was very low in both GFP-treated and DN-Pim-1-treated balloon-injured rat carotid arteries; the number of apoptotic cells was not increased at all even after treatment with DN-Pim-1 (data not shown). Thus, although practically it was very difficult to perform quantitative analysis because of the very low frequency of apoptosis, it is likely that the suppression of neointima formation by DN-Pim-1 is due to the reduction of cell proliferation rather than increased apoptosis.  (Fig. 9). These results suggest that growth inhibition by DN-Pim-1 overexpression is mainly due to the suppression of cell cycle progression.

Induction of Pim-1 Expression by Oxidative Stress or Serum Stimulation in Cultured VSMCs
Pim-1 Expression in the Thickened Intima of Human Thoracic Aortas and Coronary Arteries Obtained from Individuals at Autopsy-Although the characteristics of human and rodent arterial walls is thought to be similar, we cannot exclude the possibility that Pim-1 is induced only in rodents. Thus, to determine whether Pim-1 is also induced in humans, we performed immunohistochemical staining for Pim-1 using human thoracic aortas and coronary arteries obtained from individuals at autopsy. As shown in Fig. 10, Pim-1-positive cells were observed in the thickened intima of thoracic aortas obtained from three individuals at autopsy aged 73 (case 1), 45 (case 2), and 35 years (case 3) whose causes of death were acute myocardial infarction (case 1 and case 2) and dilated cardiomyopathy (case 3). Next, to determine in which cells Pim-1 is induced, we performed double immunostaining for Pim-1 and ␣-smooth muscle actin, a marker for smooth muscle cells. As shown in Fig. 11, in the aorta with atherosclerotic change from a 73-year-old subject (case 1), Pim-1-positive cells (Fig. 11, B and C, brown) were also stained with ␣-smooth muscle actin (Fig. 11C, red). In contrast, these cells were not stained with a mouse monoclonal antibody against human CD 68, a marker for macrophages (KP1) (DAKO) (data not shown). These results suggest that Pim-1 expression is induced in VSMCs in the human aorta. In addition, Pim-1-positive cells were also observed predominantly in the thickened intima of coronary arteries obtained from three individuals at autopsy aged 62 (case 4), 83 (case 5), and 32-years (case 6) whose cause of death were acute myocardial infarction, aortic aneurysm, and primary pulmonary hypertension, respectively (Fig. 12). Thus, Pim-1 expression was observed in human arteries showing intimal thickening as well as in the neointima of balloon-injured rat carotid arteries.

DISCUSSION
Injury of the arterial wall initiates the synthesis and release of numerous mitogens and the rapid induction of proto-oncogenes, leading to the proliferation of VSMC and neointima formation (1,2). In the present study, we identified Pim-1 as one of the candidate molecules involved in this process; the immunoreactivity of Pim-1 was detected in thickened intima induced by balloon injury of rat carotid arteries and in the thickened human aortas and coronary arteries, including atherosclerotic lesions. To the best of our knowledge, this is the first report showing Pim-1 expression in the neointima of balloon-injured rat carotid arteries and in human arteries showing intimal thickening. To evaluate the role of Pim-1, we prepared an adenovirus expressing a dominant negative form of the Pim-1 protein and found that the suppression of Pim-1 activity markedly attenuated neointima formation and reduced the number of PCNA-positive VSMCs in the balloon-injured rat carotid artery, indicating that DN-Pim-1 exerted potent antiproliferative effects. Furthermore, results from the cell culture experiments showed that reduction of cell proliferation by DN-Pim-1 overexpression is mainly due to the suppression of cell cycle progression, implying that this is the mechanism underlying the reduction of neointima formation in balloon-injured carotid arteries infected with Ad-DN-Pim-1. These findings suggest that Pim-1 plays a critical role in VSMC proliferation under certain conditions. Because functional analysis by RNA interference in embryonic stem cells has recently demonstrated that Pim-1 is required for their differentiation into VSMCs (26), it is likely that Pim-1 plays at least two roles in VSMCs; one is a physiological role in differentiation into VSMCs, and another is a role in VSMC proliferation under certain conditions.
It is well known that reactive oxygen species stimulate VSMC growth and expression of various proto-oncogenes (28). Although we have not studied the precise molecular mechanism of the regulation of Pim-1 expression in VSMCs, we demonstrated that Pim-1 expression in cultured VSMCs was markedly induced by oxidative stress (Fig. 5) and that the oxidative stress-mediated increases in cell number, thymidine incorporation, and cell cycle progression were clearly suppressed by DN-Pim-1 overexpression (Figs. 7-9). These results indicate that oxidative stress-mediated Pim-1 expression plays an important role in VSMC proliferation, at least under the in vitro conditions used. Thus, although not examined in this study, it would be important to clarify the mechanism as to how Pim-1 is induced by oxidative stress in VSMCs. Several signal transduction pathways including c-Jun N-terminal kinase, IB kinase, and p38 mitogen-activated protein kinase are known to be activated by oxidative stress in several cell types. Among the various kinases, inactivation of the c-Jun N-terminal kinase pathway has clearly been shown to suppress VSMC proliferation in a balloon injury model (29), and, thus, we assume that activation of the c-Jun N-terminal kinase pathway at least in part explains the oxidative stress-mediated induction of Pim-1.
Pim-1 is a serine/threonine kinase that is able to phosphorylate and regulate different proteins. Several substrates of Pim-1 have been identified in hematopoietic cells, some of which are potentially involved in cell cycle progression in VSMCs, although the substrate that mediates the proliferative effect of Pim-1 in VSMCs remains unknown. For example, it was reported that Pim-1 phosphorylates and activates Cdc25A (30), a phosphatase that promotes cell cycle progression, and c-Myb (22,23), a transcription factor that is essential for VSMC replication (7,8). These factors are likely to be involved in cell cycle progression in VSMCs and, thus, may explain the molecular mechanism for Pim-1-mediated VSMC proliferation. It was also reported that Pim-1 inactivates the cyclin-dependent kinase inhibitor p21 by phosphorylation (24), which could lead to cell cycle progression in VSMCs. Pim-1 may also phosphorylate yet unknown targets involved in the amplification of VSMCs. In addition, Pim-1 cooperates with c-Myc (20, 21), which is well known to play an important role in VSMC pro- FIG. 11. Double immunostaining for Pim-1 and ␣-smooth muscle actin. Hematoxylin-eosin staining (A), immunostaining for Pim-1 (B), and double immunostaining for Pim-1 and ␣-smooth muscle actin (C) in the consecutive sections were carried out using the aorta with atherosclerotic change from a 73year-old subject (case 1). Pim-1-positive cells (brown) (B and C) are also stained with ␣-smooth muscle actin (red color) (C). Original magnification in all panels is ϫ200.
FIG. 12. Pim-1 expression in the thickened intima of human coronary arteries. Coronary arteries were obtained at autopsy from three individuals aged 62 (case 4), 83 (case 5), and 32 years (case 6) whose causes of death were acute myocardial infarction, aortic aneurysm, and primary pulmonary hypertension, respectively. Pim-1-positive cells (brown) are observed predominantly in the thickened intima of the coronary arteries obtained from all three individuals. Counter-staining for the nucleus (blue) was carried out by Mayer's hematoxylin. Top row, original magnification ϫ100; bottom row, original magnification ϫ400.
liferation and thereby may contribute to cell cycle progression in VSMCs. Furthermore, although not examined in this study, DN-Pim-1 may also have some other beneficial effects in addition to its anti-proliferative effect. For example, there is the possibility that DN-Pim-1 inhibits the migration of VSMCs and/or the production of the extracellular matrix and, thus, contributes to the suppression of neointima formation.
As shown in this study, Pim-1 is likely to play a crucial role in VSMC proliferation, but how we can apply the present results to clinical medicine (e.g. treatment for occlusive vascular diseases) remains to be elucidated. Although adenovirus experiments have been useful for clarifying the mechanisms underlying various diseases, it would obviously be very difficult to use adenovirus treatment itself in clinical medicine because of its side effect. Thus, it will be very important in the future to investigate how we can apply the various significant data obtained to date with different kinds of adenovirus into clinical medicine. One possibility would be to develop a specific inhibitor of the target molecule. Considering all of our present data, small Pim-1-specific kinase inhibitor molecules could be useful for suppressing VSMC proliferation (neointimal thickening), which eventually leads to occlusive vascular diseases. Another possibility would be to establish new adenovirus systems that do not exert a deleterious side effect.
Taken together, Pim-1 expression was observed in the neointima of balloon-injured rat carotid arteries and in human aortas and coronary arteries showing intimal thickening, and specific inhibition of the Pim-1 function markedly suppressed neointima formation after balloon injury and the proliferation of cultured VSMCs, suggesting that Pim-1 plays a role in VSMC proliferation.